Inhibitory Effects of Novel Hydrophilic Cyclodextrin Derivatives on Nitric Oxide Production in Macrophages Stimulated with Lipopolysaccharide

Hidetoshi Arima, Yoji Nishinroto, Keiichi Motoyama, Fumitoshi Hirayama, and Kaneto Uekama*

Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan.
Received September 11, 2000; Received March 26, 2001, accepted April 29, 2001

Abstract

Purpose. The objective of this study is to examine the effects of cyclodextrins (CyDs) on nitric oxide (NO) production in macrophages stimulated with lipopolysaccharide (LPS).
Methods. RAW264.7 cells, a mouse macrophage-like cell, were used. Cytotoxicity of CyDs was evaluated by WST-1 method. Nitrite, iNOS, and iNOS mRNA were determined by Griess method, Western blotting, and reverse transcription-polymerase chain reaction (RT-PCR) analysis, respectively. The interaction of LPS with CyDs was evaluated by utilizing a competitive inclusion phenomenon. The binding of FITC-Iabeled LPS to the surface of RAW264.7 cells was measured by a flow cytometry.
Results. Of 15 CyDs, 2,6-di-O-methyl-α-CyD (DM-α-CyD), and 2,6-di-O-methyl-3-0- acetyl-β-cyclodextrin (DMA-β-CyD) had greater inhibitory activity than did the other CyDs against NO production in RAW264.7 cells stimulated with LPS, without showing any cytotoxicity. DM-α-CyD and DMA-β-CyD specifically inhibited the increase in iNOS and iNOS mRNA Ievels elicited by stimulation with LPS in RAW264.7 cells. DM-α-CyD and DMA-β-CyD suppressed the binding of FITC-Iabeled LPS to the surface of cells, probably resulting in inhibitory effects on iNOS expression and NO production. DM-α-CyD had a greater interaction with RAW264.7 cells than did DMAP-CyD. The pretreatment of RAW264.7 cells with DM-α-CyD, not DMA-β-CyD, decreased the LPS binding to the cell surface. The results suggested that the inhibitory mechanism of the LPS binding to the cell surface is different between DM-α-CyD and DMA-β-CyD. Conclusions. The present results suggest that DM-α-CyD and DMA-β-CyD attenuates NO production by inhibiting iNOS gene expression in RAW264.7 cells stimulated with LPS, probably due to the suppression of LPS binding to LPS receptors on the cells in the different way.
KEY WORDS: cyclodextrin; nitric oxide; Iipopolysaccharide; macrophages; inducible nitric oxide synthase.